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Title: Arabidopsis UDP-sugar pyrophosphorylase: evidence for two isoforms

item Gronwald, John
item Miller, Susan
item Vance, Carroll

Submitted to: Plant Physiology and Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/11/2008
Publication Date: 11/7/2008
Citation: Gronwald, J.W., Miller, S.S., Vance, C.P. 2008. Arabidopsis UDP-sugar pyrophosphorylase: evidence for two isoforms. Plant Physiology and Biochemistry. 46(12):1101-1105.

Interpretive Summary: Alfalfa, the largest forage legume crop in the U.S., is important because of its high feed value for livestock and because it has considerable potential for development as a bioenergy crop. For both of these uses, a modification of the cell walls of alfalfa stems is desirable. Pectin is a matrix polysaccharide found in plant cell walls. An increase in pectin in stem cell walls would improve alfalfa's value as a livestock feed while decreasing pectin would improve its value as a bioenergy crop. In order to develop new alfalfa varieties with modified stem cell walls, a better understanding of the metabolic pathways that regulate biosynthesis of pectin and other cell wall matrix polysaccharides is needed. The myo-inositiol oxidation (MIO) pathway plays a key role in the synthesis of cell wall matrix polysaccharides in plants. The last step in this pathway involves the conversion of a sugar (glucuronic acid-1-P) to its activated form (UDP-glucuronic acid). In earlier research conducted with the model plant Arabidopsis, we identified the USP (UDP-sugar pyrophosphorylase) gene which makes a protein that converts glucuronic acid-1-P to UDP-glucuronic acid. These results suggested that USP protein catalyzes the last step in the MIO pathway. However, it was not clear whether USP is the only protein in plants that can catalyze this reaction. In order to determine this, we make a specific tag molecule (antibody) to the Arabidopsis USP protein. The antibody binds specifically to USP protein and removes it from plant cell extracts. If USP is the only protein that is able to convert glucuronic acid-1-P to UDP-glucuronic acid, then its removal by the antibody will eliminate this reaction in plant extracts. Our results confirmed our hypothesis that USP is the only protein in plant extracts that is able to convert glucuronic acid-1-P to its activated form. Our research using the antibody also showed that the USP gene in Arabidopsis produces two slightly different versions (isoforms) of the USP protein which exhibit small differences in molecular weight. The results of this research indicate that USP plays a key role in the MIO pathway. Modifying the expression of this gene may allow for desirable changes to be made in pectin content of cell walls. The new knowledge gained in this study will be used in ongoing research to modify cell walls in alfalfa stems and increase the value of alfalfa as a livestock feed and bioenergy crop.

Technical Abstract: Arabidopsis UDP-sugar pyrophosphorylase (AtUSP, EC is a broad substrate pyrophosphorylase that exhibits activity with GlcA-1-P, Gal-1-P, and Glc-1-P. Immunoblots using polyclonal antibodies raised to recombinant AtUSP demonstrated the presence of two USP isoforms of approximately 70 kDa (USP1) and 66 kDa (USP2) in crude extracts of Arabidopsis tissues. The appearance of the 66 kDa isoform was not due to proteolytic cleavage of USP1 during extraction. Trypsin digestion of bands on SDS gels corresponding to the location of the two isoforms followed by tandem mass spectrometry confirmed that USP was present in both bands. Both USP isoforms were located in the cytosol as determined by immunoblots of cellular fractions obtained by differential centrifugation. However, USP1 was also detected in the microsomal fraction. Immunoprecipitation assays demonstrated that AtUSP antibodies removed USP activity measured in floret extracts. These results, indicating that USP is the only pyrophosphorylase that utilizes UDP-GlcA as a substrate, suggest that it serves as the terminal enzyme of the myo-inositol oxidation pathway.